LTA4 , hydrolase inhibitors

ABSTRACT

The present invention provides compounds having the structure: ##STR1## and pharmaceutically acceptable salts and stereoisomers thereof that are useful in the treatment of inflammatory diseases which are mediated by LTB 4  production, such as psoriasis, ulcerative colitis, IBD, and asthma.

FIELD OF THE INVENTION

This invention relates generally to anti-inflammatory compounds andpharmaceutical compositions, and more particularly to anti-inflammatorycompounds and compositions which are capable of inhibiting leukotrieneA₄ hydrolase.

BACKGROUND OF THE INVENTION

LTA₄ hydrolase is a requisite enzyme in the biosynthetic pathway leadingto LTB₄ formation. LTB₄ is a proinflammatory compound. R. Lewis, et al.,N. Engl. J. Med. 323, 645-655 (1990) have demonstrated that LTB₄ is apotent granulocyte agonist inducing chemotaxis, aggregation,degranulation, adherence and priming of inflammatory cells for inductionby other agonists. Binding of LTB₄ to receptors is stereospecific withtwo distinct classes of binding sites. A. Lin, et al., Prostaglandins28, 837-849 (1984). A high affinity site 4-5×10⁻¹⁰ M! mediateschemotaxis and chemokinesis while lower affinity sites 0.6-5×10⁻⁷ M!stimulate granular secretion and oxidative burst. The LTB₄ receptor isassociated with a GTP-binding protein that regulates affinity andtransduces signals. T. Schepers, et al., J. Biol. Chem. 267, 159-165(1992). Elevated LTB₄ levels have been reported for many diseases. Mostprominently, elevated LTB₄ levels have been correlated to the pathologyof inflammatory bowel disease (IBD) including Crohn's disease andulcerative colitis and in psoriasis. P. Sharon, et al., Gastroent. 86,453-460; K. Lauritsen, et al., Gastroent. 95, 11-17 (1989); S. Brain, etal., Br. J. Pharm., 83, 313-317 (1984). Other properties of LTB₄ whichmay contribute to disease processes are: stimulation of mucus secretion;stimulation of cytokine production; and the ability to actsynergistically with other inflammatory mediators such as prostaglandinsand cysteinyl leukotrienes thereby amplifying the inflammatory process.

B. Samuelsson, et al., J. Biol Chem., 264, 19469-19472 (1989) have shownthat LTB₄ biosynthesis from arachidonic acid involves the action of 2enzymes, 5-lipoxygenase 5-LO! and LTA₄ hydrolase. 5-LO transformsarachidonic acid to 5-HPETE and subsequent formation of LTA₄, which isan unstable allylic epoxide intermediate which is enzymaticallyhydrolyzed by LTA₄ hydrolase to form the dihydroxy acid LTB₄.

LTA₄ hydrolase is distinct from cytosolic and microsomal epoxidehydrolases based on strict substrate requirements, product formation5(S),12(R) vs. 5(S),6(R)! for mouse liver cytosolic epoxide hydrolase,and lack of inhibition by inhibitors of cytosolic epoxide hydrolase.LTA₄ hydrolase appears to be ubiquitously distributed in mammaliantissues even in cell types that do not express 5-LO, suggesting theimportance of transcellular metabolism of LTA₄. While peptidomimeticcompounds such as bestatin and captopril have been shown to exhibit LTA₄hydrolase inhibitory activity, they are not able to satisfy therequirement of a small organic compound which is capable of cellularpenetration. It would therefore be very advantageous to be able toprovide low molecular weight inhibitors of LTB₄ biosynthesis whichpreferably exhibit oral activity in vivo at desirably lowconcentrations.

SUMMARY OF THE INVENTION

Applicants have now discovered that compounds having the structure:##STR2## and pharmaceutically acceptable salts and stereoisomers thereofpossess LTA₄ hydrolase inhibitor activity wherein ##STR3## wherein .....represents a single or double bond

q is 1 or 2, and

Y is --O--, --S--, --CH₂ --, or --CH--

B is --O--, --CH₂ -- or --CH₂ O--

n is an integer from 2 to 4

R¹ is H or C₁ to C₄ alkyl

R² is (CH₂)_(m) R³ wherein m is an integer from 1 to 3

R³ is CO₂ R⁴

R⁴ is H alkyl, amino, alkylamino, dialkylamino

or NR¹ R² is combined to form ##STR4## wherein r is 1 or 2, p is 0 to 3and R³ is as defined above.

DETAILED DESCRIPTION

In one of its embodiments, the present invention entails compoundshaving the structure: ##STR5## and pharmaceutically acceptable salts andstereoisomers thereof, wherein A, B, R¹, R², and n are as defined above.

The compounds of the present invention, in several embodiments, maycomprise a carboxylic acid or ester moiety. It will be appreciated bythose of ordinary skill in the art that a compound of the presentinvention comprising an ester moiety is readily converted, in vivo,especially when administered orally, into its corresponding carboxylicacid form. The ester-containing compounds of the present invention aretherefore prodrugs of their carboxylic acid form.

In another of its aspects, the invention entails pharmaceuticalcomposition comprising a pharmacologically effective amount of at leastone of the compounds defined above and a pharmaceutically acceptablecarrier.

In still another of its embodiments the present invention involves amethod for treating a mammal exhibiting an LTB4 mediated inflammatorycondition comprising administering to the mammal a pharmacologicallyeffective amount of a compound of the invention.

The term "lower alkyl" means straight or branched chain alkyl having 1to 6 carbon atoms such as methyl, ethyl, propyl, butyl, pentyl, hexyland the branched chain isomers thereof. The term "lower alkoxy" meansstraight or branched chain alkoxy having 1 to 6 carbon atoms such asmethoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy and the branched chainisomers thereof. The term "allyl" as used herein means the 1-propenylradical, --CH₂ --CH₂ ═CH₂. The term "halo" or "halogen" means fluoro,chloro, bromo, or iodo.

Included within the classes and subclasses of compounds embraced by thisinvention are isomeric forms of the described compounds includingdiastereoisomers, enantiomers and tautomeric forms of the describedcompounds. Pharmaceutically acceptable salts of such compounds are alsoincluded as well as pharmaceutically acceptable salts of such isomersand tautomers.

In the structures disclosed herein a bond drawn across a bond in a ringindicates that the bond can be to any available atom of the ringstructure.

The expression "pharmaceutically acceptable salts" is intended toinclude those salts capable of being formed with the compounds of thepresent invention without materially altering the chemical structure orpharmacological properties thereof. Such salts can be inorganic andorganic cations or acid addition salts, including, but not limited tosodium, potassium, calcium, ammonium, alkylammonium, quaternaryammonium, triethanolamine, lysine, hydrochloride, hydrobromide, andothers well known to those of ordinary skill in the art. The foregoingsalts are prepared in the conventional manner by neutralization of thecompounds of this invention with the desired base or acid.

The compounds of the present invention can be administered to a subjectin such oral dosage forms as tablets, capsules, pills, powders,granules, elixirs or syrups, as well as aerosols for inhalation.Likewise, administration may be effected intravascularly,subcutaneously, or intramuscularly using dosage forms known to those ofordinary skill in the pharmaceutical arts. In general, the preferredform of administration is oral. An effective but non-toxic amount of thecompound is employed in treatment. The dosage regimen utilizing thepresent compounds is selected in accordance with a variety of factorsincluding the type, age, weight, sex and medical condition of thepatient; the severity of the condition to be ameliorated; and the routeof administration. A physician of ordinary skill can readily determineand prescribe a "pharmaceutically effective amount" of at least one ofthe compounds defined above, that is, the effective amount of thecompound required to prevent, treat or arrest the progress of thecondition. Dosages of the compounds of the present invention will rangegenerally between 0.1 mg/kg/day to about 100 mg/kg/day and preferablybetween about 0.5 mg/kg/day to about 50 mg/kg/day when administered tosubjects suffering from allergic or hypersensitivity reactions orinflammation. The compounds may also be administered transdermally ortopically to treat proliferative skin conditions such as psoriasis. Thedaily dosage may be administered in a single dose or in equal divideddoses, for example, three to four times daily. The subject is typicallya mammal and, in particular, a human patient.

As used herein the phrase "LTA₄ hydrolase inhibitor" means a compoundthat is capable of exhibiting an IC₅₀ of less than 1 mM in an in vitroassay employing 10 μg/ml of LTA₄ hydrolase enzyme (specific activity 600nMoles LTB₄ /min/mg of enzyme) in the presence of 25 μM substrate (LTA₄)in a total reaction volume of 100 μl.

In the pharmaceutical compositions and methods of the present invention,at least one of the active compounds of the invention or apharmaceutically acceptable salt thereof will typically be administeredin admixture with suitable pharmaceutical diluents, excipients orcarriers (collectively referred to herein as "carrier materials")suitably selected with respect to the intended form of administrationand consistent with conventional pharmaceutical practices. For example,the pharmaceutical compositions of this inventio ncan be administered asoral tablets, capsules, elixirs, syrups and the like For oraladministration in the form of tablets or capsules, the active drugcomponent may be combined with any oral non-toxic pharmaceuticallyacceptable inert carrier such as lactose, starch, sucrose, cellulose,magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol andthe like; for oral administration in liquid form, the active drugcomponent may be combined with any oral non-toxic pharmaceuticallyacceptable inert carrier such as ethanol and the like. Moreover, whendesired or necessary, suitable binders, lubricants, disintegratingagents and coloring agents can also be incorporated in the mixture.Suitable binders include starch, gelatin, natural sugars, cornsweeteners, natural and synthetic gums such as acacia, sodium alginate,carboxymethylcellulose, polyethylene glycol and waxes. Lubricants foruse in these dosage forms include boric acid, sodium benzoate, sodiumacetate, sodium chloride and the like. Disintegrators include, withoutlimitation, starch, methylcellulose, agar, bentonite, guar gum and thelike.

By virtue of their activity as LTA₄ hydrolase inhibitors, the compoundsof the invention are useful in treating inflammatory conditions mediatedby LTB₄ production in mammals such as psoriasis, contact and atrophicdermatitis, Crohn's disease, ulcerative colitis, inflammatory boweldisease, multiple sclerosis, ankylosing spondylitis, arthritis, asthmaand the like. Similarly, the compounds of the invention can be used inpreventing recurring inflammatory attacks. A physician or veterinarianof ordinary skill can readily determine whether a subject exhibits theinflammatory condition. A preferred utility relates to treatment ofulcerative colitis.

The compounds of the invention are prepared from readily availablestarting materials by any of the following alternate processes in aconventional manner. The following reaction schemes describe methodswhich can be employed for preparing the compounds of the inventionincluding starting materials, intermediates and reaction conditions. Thefollowing terms, as used herein, have the following definitions:

    ______________________________________    NMMO          N-methylmorpholine-N-oxide    Me            methyl    SitBuMe.sub.2 t-butyldimethylsilyl    nBuLi         n-butyllithium    THF           tetrahydrofuran    Et.sub.2 O    diethyl ether    EtOH          ethyl alcohol    Pd/C          palladium on carbon    TFA           trifluoroacetic acid    Et.sub.3 SiH  triethylsilane    TBAF          tetrabutylammonium fluoride    DMF           dimethylformamide    nBu.sub.4 NBr tetra-n-butylammonium bromide    TsCl          tosylchloride or p-toluenesulfonyl-                  chloride    TsO           tosylate or p-toluenesulfonate    MeOH          methyl alcohol    AcOH          acetic acid    Bn            benzyl    DEAD          diethylazodicarboxylate    Ph.sub.3 P    triphenylphosphine    MCPBA         metachloroperbenzoic acid    LAH           lithium aluminum hydride    TsOH          tosic acid or p-toluenesulfonic acid    LDA           lithium diisopropylamide    DSC           disuccinylcarbonate    nBuOH         n-butyl alcohol    TFAA          trifluoroacetic anhydride    Me.sub.3 SnN.sub.3                  trimethyl-tin azide    TMS           trimethyl silyl    Ac.sub.2 O    acetic anhydride    Ac            acetate    EtOAc         ethyl acetate    Hep           heptane    ______________________________________

    __________________________________________________________________________    General Scheme    __________________________________________________________________________                    1 #STR6##    2 #STR7##    3 #STR8##    4 #STR9##    5 #STR10##    6 #STR11##              7 #STR12##           8 #STR13##    9 #STR14##    0 #STR15##    a)    KOH, Cu°, 160° C.-200° C.    b) ZnCl.sub.2, ethanolamine, 130° C.    c) NiO.sub.2, benzene, reflux    d) CH.sub.2 Cl.sub.2, BBr.sub.3, -78° C.    e) Ethylene carbonate, DMF, nBu.sub.4 NBr, 140° C.    f) TsCl, pyridine, CH.sub.2 Cl.sub.2, 0° C.    g) DMF, K.sub.2 CO.sub.3, ZH, where Z is NR.sup.1 R.sup.2 wherein R.sup.1    and    R.sup.2 are as defined hereinbefore    h)    1 #STR16##    i)    2 #STR17##    j) CH.sub.3 CN, H.sub.2 NR', 55° C.    k) CH.sub.2 Cl.sub.2, methylacrylate, room temp.    l)    3 #STR18##    m) HCl    n) KOH, DMSO, tBuOH, reflux    o) Lawesson's reagent, toluene, reflux    p) (CO.sub.2 H).sub.2, ClCH.sub.2 CH(OMe).sub.2, reflux    __________________________________________________________________________

EXAMPLE 1 ##STR19##

(a) A mixture of 4-iodobenzonitrile (5.06 g, 22 mmol), 4-methoxyphenol(2.72 g, 22 mmol), potassium carbonate (3.182 g, 22 mmol), and copperbronze (1.39, 22 mmol) in pyridine (120 ml) was heated to reflux underargon for 4 days. The reaction was allowed to cool to room temperatureand concentrated in vacuo. The brown residue was acidified to pH=1 withconcentrated HCl and diluted with water. The mixture was extracted withEtOAc (2X) and the organic layers collected. The organic layer was driedover MgSO₄ and concentrated in vacuo to give a black/brown solid (4.56g). The solid was purified by column chromatography (5% EtOAc/hexanefollowed by 10% EtOAc/hexane) to give a white solid (1.8 g). NMRspectrum is consistent with structure (a) above. ##STR20##

(b) A mixture of fused ZnCl₂ (0.782 g, 5.3 mmol), the compound from stepa (0.548 g, 2.2 mmol), and ethanolamine (15 ml) was heated to 130-140°C. for 4 hours The reaction was diluted with CH₂ Cl₂, and washed withwater (2X) and brine. The organic layer was collected and dried overMgSO₄. Concentration in vacuo gave a white solid (0.71 g). The solid waspurified by column chromatography (100 g silica gel, 5% MeOH/CH₂ Cl₂(500 ml)) gave the desired product as a white solid (0.29 g). NMRspectrum is consistent with structure (b) above. ##STR21##

(c) A mixture of the compound of step b (0.149 g, 0.59 mmol) and NiO₂(0.838 g, 8.9 mmol) in benzene (10 ml) was heated to reflux for 17hours. The reaction was allowed to cool to room temperature and filteredthrough celite. Concentration of the filtrate gave a white solid (0.10g). NMR spectrum is consistent with structure (c) above.

EXAMPLE 2 ##STR22##

A mixture of 4-cyanophenol (1.856 g, 15.4 mmol) and potassium hydroxide(0.868 g, 14.1 mmol) was heated to 140° C. under argon. The resultingsolution resolidified within 15 min. of heating. At this time,4-iodoanisole (3.039 g, 12.8 mmol) was added followed by activated Cu(0.277 g) and the reaction mixture was heated to 170° C. for 20 hours.The reaction was allowed to cool to room temperature and 10% NaOH added.The mixture was extracted with Et₂ O (4×75 ml). The organic layers werecollected, washed with brine and dried over MgSO₄. Concentration invacuo gave a red/brown oil (0.68 g). The oil was purified by columnchromatography (50 g silica gel; 5% EtOAc/hexane followed by 10%EtOAc/hexane) to give the product as a pale yellow solid (0.140 g). NMRspectrum is consistent with the structure above.

EXAMPLE 3 ##STR23##

The procedures described in Example 2 were repeated using 4-phenylphenol(4.366 g, 25.6 mmol) in place of 4-cyanophenol, and 4-iodoanisole(5.053, g, 21.4 mmol). The reaction was heated to 200° C. for 3.5 hours.After work-up, a pale yellow solid was collected. The solid wasrecrystallized from MeOH to give the desired product (1.03 g). NMRspectrum is consistent with the structure above.

EXAMPLE 4 ##STR24##

A solution of the compound of Example 1 (0.08 g, 0.3 mmol) in CH₂ Cl₂ (2Ml) was cooled to -78° C. A 1 M solution of BBr₃ in CH₂ Cl₂ (0.66 ml)was added slowly under argon. The reaction was allowed to warm slowly toroom temperature over 1.5 hours. The reaction was concentrated in vacuoand a mixture of water and CH₂ Cl₂ was added to the residue. The organiclayer was collected and washed with brine. Concentration in vacuo gave abrown oil (0.079 g). The oil solidified upon standing at roomtemperature. The solid was slurried with CH₂ Cl₂ (3-5 ml) and theundissolved solid was collected by vacuum filtration to give a greysolid (0.047 g). NMR spectrum is consistent with the structure above.

EXAMPLE 5 ##STR25##

The procedures described in Example 4 were repeated using the compoundof Example 2 (0.259 g, 1.2 mmol) in place of the compound of Example 1.After work-up, a blue solid was obtained as the desired product (0.262g). NMR spectrum is consistent with the structure above.

EXAMPLE 6 ##STR26##

The procedures described in Example 4 were repeated using the compoundof Example 3 (1.03 g, 3.7 mmol) in place of the compound of Example 1.Upon work-up, the desired product was obtained as a white solid (0.887g). NMR spectrum is consistent with the structure above.

EXAMPLE 7 ##STR27##

A mixture of the compound of Example 4 (0.04 g, 0.16 mmol), potassiumcarbonate (0.120 g, 0.79 mmol), and 1-(2-chloroethyl)pyrrolidinehydrochloride (0.037 g, 0.19 mmol) in DMF (3 ml) was heated to 80° C.(bath). After 21 hours of heating, the reaction was allowed to cool toroom temperature and diluted with EtOAc (20 ml). The resulting solutionwas washed with water (2×20 ml) and brine (20 ml). The organic layer wascollected, dried over MgSO₄, and concentrated in vacuo to give awhite/yellow solid (0.04 g). The solid was purified by platechromatography (5% MeOH/CH₂ Cl₂) to give the desired product as a tansolid (0.022 g). Anal. calc'd for C₂₁ H₂₂ N₂ O₃ +0.2H₂ O: C, 71.25; H,6.38; N, 7.91. Found: C, 71.03; H, 5.98; N, 7.80. M⁺ =350.

EXAMPLE 8 ##STR28##

The procedures described in Example 7 were repeated using the compoundof Example 6 (0.360 g, 1.4 mmol) in place of the compound of Example 4.After work-up, a yellow/white solid was obtained. The solid was furtherpurified by slurrying with MeOH to give a cream-colored solid as thedesired product (0.202 g). Anal. calc'd for C₂₄ H₂₅ NO₂ +0.2 H₂ O: C,79.40; H, 7.05; N, 3.86. Found: C, 79.65; H, 7.11; N, 3.84. MH⁺ =360.

EXAMPLE 9 ##STR29##

The procedures described in Example 7 were repeated using the compoundof Example 5 (0.262 g, 1.2 mmol) in place of the compound of Example 4.After work-up, a yellow/brown oil was obtained as the desired product(0.260 g). NMR spectrum is consistent with the structure above.

EXAMPLE 10 ##STR30##

The procedures described in step b of Example 1 were repeated using thecompound of Example 9 (0.087 g, 0.28 mmol) in place of the compound ofExample 1 step a. After work-up, a yellow/white solid was obtained. Thesolid was further purified by column chromatography (95 CHCl₃ : 5 EtOH:0.5 NH₄ OH) to give the desired product as a white solid (0.06 g). Anal.calc'd for C₂₁ H₂₄ N₂ O₃ +0.3 H₂ O: C, 70.49; H, 6.93; N, 7.83. Found:C, 70.48; H, 7.02; N, 7.77. MH⁺ =353.

EXAMPLE 11 ##STR31##

(a) To a solution of the compound of Example 6 (0.411 g, 1.6 mmol) inDMF (5 ml) was added ethylene carbonate (0.255 g, 2.9 mmol) and nBu₄ NBr(0.108 g, 0.31 mmol) under argon. The reaction was heated to 140-150° C.(bath). After 8 hours, additional ethylene carbonate (0.041 g) was addedto the reaction. The reaction was stirred at 140-150° C. for anadditional 16 hours before concentrating the reaction in vacuo. Theresulting residue was dissolved in CH₂ Cl₂ and washed with brine. Theorganic layer was collected, dried over MgSO₄, and concentrated in vacuoto give a light tan solid (0.570 g). The solid was recrystallized fromEtOAc to give the desired product (0.210 g). NMR spectrum is consistentwith the structure (a) above. ##STR32##

(b) A mixture of the compound from step a (0.085 g, 0.28 mmol) and TsCl(0.074 g, 0.36 mmol) in CH₃ CN (1.5 ml) was cooled to 0° C.Triethylamine (0.15 ml, 1.1 mmol) was then added neat under argon. Thereaction was allowed to stir at 0° C. for 5 min. before removing the icebath. The reaction was stirred at room temperature for 22 hours and thenquenched with water. The resulting mixture was filtered and the desiredproduct was collected as a tan solid. The solid was rinsed with CH₃CN/water (3:7) then allowed to air dry to give 0.107 g. The product wascombined with 5 other previous runs and purified by columnchromatography (3:1 hexane/EtOAc) to give the desired product as a whitesolid (0.142 g). NMR spectrum is consistent with the structure (b)above. ##STR33##

(c) To a solution of the compound from step b (0.142 g, 0.3 mmol) in DMF(1.5 ml) was added ethyl isonipecotate (0.05 ml, 0.3 mmol) followed bypotassium carbonate (0.220 g, 1.5 mmol). The reaction mixture was heatedto 80° C. (bath) under argon for 19.5 hours. The reaction wasconcentrated in vacuo and the residue was diluted with water (20 ml).The mixture was extracted with EtOAc (2×35 ml). The organic layers werecombined, washed with brine, and dried over MgSO₄. Concentration invacuo gave a white/yellow solid (0.134 g). The solid was purified bycolumn chromatography (50 g silica gel, 1:1 EtOAc/hexane followed by 2:1EtOAc/hexane) to give the desired product as a white solid (0.063 g).Anal. calc'd for C₂₈ H₃₁ NO₄ : C, 75.48; H, 7.01; N, 3.14. Found: C,75.28; H, 7.07; N, 3.09. M⁺ =445.

EXAMPLE 12 ##STR34##

To a solution of the compound of Example 11 in distilled THF (5 ml) wasadded 6 M HCl. The reaction was heated to 85-95° C. for 2 hours. Thereaction was then concentrated in vacuo to give a white solid. The solidwas purified by slurrying with ether. A white solid was collected byvacuum filtration (0.014 g). Anal. calc'd for C₂₄ H₂₅ NO₂ +3.0 HCl+1.0H₂ O: C, 57.31; H, 5.92; N, 2.57. Found: C, 57.37; H, 6.02; N, 2.25. M⁺=417.

EXAMPLE 13 ##STR35##

(a) To a solution of the compound of Example 6 (0.350 g, 1.3 mmol) inmethyl ethyl ketone (4 ml) was added potassium carbonate (0.937 g, 6.7mmol) followed by bromochloropropane (0.13 ml, 1.3 mmol) under argon.The resulting mixture was heated to 85-90° C. (bath) for 21.5 hours,then heated to 95° C. for 1.5 hours. The reaction was poured into aseparatory funnel containing water (25 ml) and extracted with EtOAc(2×40 ml). The organic layers were combined, washed with brine, driedover MgSO₄, and concentrated in vacuo to give a yellow/white solid(0.442 g). The solid was purified by column chromatography (75 g silicagel, 5:1 hexane/EtOAC (500 ml)) to give the desired product as a whitesolid (0.324 g). NMR spectrum is consistent with structure (a) above.##STR36##

(b) To a mixture of the compound from step a (0.324 g, 0.96 mmol) in CH₃CN (6 ml) was added H₂ NMe (8 ml, 95.6 mmol). Upon addition of H₂ NMe, awhite solid precipitated out of the mixture. The mixture was heated to55° C. (bath) for 8.5 hours. At this time, additional H₂ NMe (2 ml) wasadded to the reaction. The reaction was stirred for another 16 hours atroom temperature then heated to 55° C. for 3 hours. The reaction wasconcentrated in vacuo and extracted with EtOAc (2×20 ml). The organiclayer was collected, cooled to 0° C., and acidified to pH 1 with 6 MHCl. At this point, no solid was observed to precipitate out ofsolution. The solution was therefore basified to pH 12 with 10% NaOH andextracted with EtOAc (2×50 ml). The organic layer was collected anddried over MgSO₄. Concentration in vacuo gave a white solid. The solidwas slurried with EtOAc and collected by vacuum filtration as thedesired product (0.224 g). NMR spectrum is consistent with the structure(b) above. ##STR37##

(c) To a solution of the compound of step b (0.224 g, 0.70 mmol) in CH₂Cl₂ (2 ml) was added methyl acrylate (0.08 ml, 0.91 mmol). The reactionwas stirred at room temperature over 48 hours. At this time, additionalmethyl acrylate was added (0.04 ml) to the reaction. The reaction wasstirred for another 3 hours, then concentrated under a stream of N₂ togive a white/yellow solid. The solid was purified by columnchromatography (50 g silica gel, 10% MeOH/CH₂ Cl₂ to give the desiredproduct as a white solid (0.200 g). Anal. calc'd for C₂₆ H₂₉ NO₄ : C,74.44; H, 6.97; N, 3.34. Found: C, 74.11; H, 6.85; N, 3.21. M⁺ =419.

EXAMPLE 14 ##STR38##

The compound of Example 13 (0.1 g) was treated with 6 M HCl under thesame reaction conditions as those described in Example 12 to give thedesired product as a white solid (0.073 g). C₂₅ H₂₈ NO₄ +1.0 HCl+0.8 H₂O: C, 65.80; H, 6.54; N, 3.07. Found: C, 65.71; H, 6.25; N, 2.81. MH⁺=406.

EXAMPLE 15 ##STR39##

200 mL of 2N ethylamine in methanol (0.4 mol), 10 mL (0.15 mol)acrylonitrile and 35 mL (0.25 mol) triethylamine were stirred in 100 mLmethanol at 25° C. for 21 hours. The mixture was concentrated and usedwithout further purification. This was stirred in 70 mL DMF with 44 mL(0.44 mol) 1-bromo-3-chloropropane and 25 mL (0.18 mol) triethylamine at40° C. for 5 hours and at 25° C. for 15 hours. The mixture was pouredinto water and ether and the ether layer was washed with 2N HCl. Theacid layer was washed with ether, made basic (>pH 10) with 45% KOH andextracted twice with ether. The ether extracts were dried over Na₂ SO₄and concentrated to provide the desired compound (21.7 g, 0.124 mol) asa colorless oil: ¹ H NMR (CDCl₃) δ1.04 (t,3H), 1.88 (m, 2H), 2.40-2.65(m, 6H), 3.65 (t, 2H).

EXAMPLE 16 ##STR40##

The compound was prepared as described for Example 15, using a solutionof methylamine in place of ethylamine.

EXAMPLE 17 ##STR41##

(a) To a suspension of the product from Example 2 (10.0 g, 44 mmol) int-butanol (80 mL) was added 30 mL dimethylsulfoxide (DMSO) and powderedKOH (9.1 g, 162 mmol). The mixture was heated at reflux for 2 hours. Themixture was cooled and diluted with water (100 mL). The white solidprecipitate was collected by filtration and washed with water (4×150mL). The solid was dried in vacuo to give 9.6 g (89%) of (a): mp195-196° C. ##STR42##

(b) To a suspension of the compound from step (a) (4.2 g, 17.3 mmol) intoluene (80 mL) was added Lawesson's reagent (7.0 g, 17.3 mmol). Themixture was heated at reflux for 3 hours, cooled and concentrated invacuo. The residue was chromatographed on silica gel (1:1 hexane/ethylacetate) to give 2.2 g (49%) of (b). ##STR43##

(c) A mixture of oxalic acid (590 mg, 6.5 mmol) and chloroacetaldehydedimethyl acetal (0.75 mL, 6.5 mmol) was heated at reflux for 1 hour. Theoil bath was removed for 10 minutes and the compound from step (b) (1.7g, 6.5 mmol) was added. The resulting mixture was heated at reflux for 2hours. The mixture was cooled to room temperature and 30% HCl (3.5 mL)was added. The mixture was heated at reflux for 10 minutes, cooled anddiluted with water. The reaction mixture was extracted with CH₂ Cl₂(3×10 mL). The organic solution was dried (Na₂ SO₄) and concentrated invacuo. The residue was chromatographed (3:1 hexane/ethyl acetate) togive the 925 mg (50%) of (c) as a crystalline solid: mp 92-93° C.; Anal.calcd for C₁₆ H₁₃ NO₂ S: C, 67.82; H, 4.62; N, 4.94. Found: C, 67.66; H,4.50; N, 4.86. ##STR44##

(d) To a solution of the compound from step (c) (280 mg, 1.0 mmol) inCH₂ Cl₂ (3 mL) at -78° C. was added boron tribromide (1.8 mL of a 1Msolution in CH₂ Cl₂). The solution was kept at -78° C. for 1 h and thenwarmed to room temperature over 2 hours. The reaction solution wasdiluted with water and extracted with CH₂ Cl₂ (2×20 mL). The combinedorganic solution was dried (Na₂ SO₄) and concentrated in vacuo to give210 mg (78%) of (d). ##STR45##

(e) To a suspension of powdered KOH (63 mg, 1.1 mmol) in DMSO (1 mL) asadded via canula a solution of the compound from step (d) (200 mg, 0.74mmol) in DMSO (2 mL). The mixture was stirred at room temperature for 5min and the product of Example 16 (118 mg, 0.74 mmol) in DMSO (1 mL) wasadded via canula. The reaction mixture was heated at 45° C. for 4 hours.The mixture was cooled to room temperature and partitioned between waterand ether (15 mL). The aqueous solution was extracted with ether (2×10mL). The combined organic solution was dried (Na₂ SO₄) and concentratedin vacuo. The residue was chromatographed (ethyl acetate) to give 112 mg(39%) of (e) as a crystalline solid: mp 63-64° C. ##STR46##

(f) A solution of the compound from step (e) (100 mg, 0.25 mmol) in 6NHCl (2 mL) was heated at 90° C. for 17 hours. The solution was cooled toroom temperature and brought to pH 8 with 10% NaOH. The aqueous solutionwas extracted with CH₂ Cl₂ (3×15 mL). The organic solution wasconcentrated in vacuo. The residue as chromatographed on silica (85:14:1CH₂ Cl₂ /MeOH/NH₄ OH) to give 40 mg (39%) of (f) as a crystalline solid:mp 143-144° C.

EXAMPLE 18 ##STR47##

The product from Example 4 (434 mg, 1.7 mmol), the product from Example16 (305 mg, 1.9 mmol) and powdered KOH (158 mg, 2.8 mmol) were stirredin 20 mL DMF at 50° C. for 12 hours. The mixture was cooled and dilutedwith 75 mL H₂ O. The aqueous base was separated and extracted with 3×25mL methyl t-butyl ether (MTBE). The combined organic phases were dried(MgSO₄) and concentrated to afford the crude product as a brown oil. Thecrude nitrile was dissolved in 5 mL MTBE and 5 mL concentrated HCl wasadded. The MTBE was distilled from the reaction and an additional 2 mLconcentrated HCl was added. The reaction was heated to 95° C. for 24hours. After cooling, the mixture was diluted with 50 mL H₂ O andneutralized with a saturated NaHCO₃ solution. The aqueous phase wasextracted with 4×15 mL CH₂ Cl₂ and the extracts dried (MgSO₄) andconcentrated to afford a yellow oil. The oil was dissolved in 1 mLmethanol and 3M ethanolic HCl was added until a precipitate formed. Thetan solid was filtered and dried: Anal. calcd for C₂₂ H₂₄ N₂ O₅ 1.5HCL1.0 H₂ O: C. 56.32; H, 5.91; N, 5.97; Cl, 11.34. Found: C, 56.61; H,5.75; N, 5.32; Cl, 11.55.

EXAMPLE 19 ##STR48##

The above compound was prepared in the same manner as Example 18substituting the product from Example 15 in place of the product ofExample 16. The HCl salt was isolated as a tan solid: Anal. calcd forC₂₃ H₂₆ N₂ O₅ 1.25 HCl1.0 H₂ O: C, 58.27; H, 6.22; N, 5.91; Cl, 9.35.Found: C, 58.16; H, 6.25; N, 5.26; Cl, 9.14.

LTA Hydrolase Methods

The following Table presents data demonstrating the pharmacologicalactivity of the LTA hydrolase inhibitors of the present invention. Oneor more of three different assays, (1) an in vitro LTA hydrolase enzymeassay, (2) a human whole blood assay utilizing calcium ionophorestimulation, and (3) a murine ex vivo assay utilizing calcium ionophorestimulation were employed to determine the level of LTA hydrolaseinhibitor activity.

Recombinant Human LTA Hydrolase Assay for LTA Hydrolase InhibitorActivity

Compounds of the present invention were tested for LTA hydrolaseinhibitor activity against recombinant human LTA hydrolase (rhLTAH).Recombinant human LTA hydrolase-encoding vectors were prepared and usedto express rhLTAH essentially as described by J. Gierse, et al., ProteinExpression and Purification, 4, 358-366 (1993). Briefly, LTA hydrolaseencoding DNA was amplified by polymerase chain reaction using a pair ofoligonucleotide primers based on the nucleotide sequence from the5'-end, and the complement of the 3'-end, of the coding region of theLTA hydrolase gene, the nucleotide sequence of which gene is known.(See, C. Funk, et al., Proc. Natl. Acad. Sci. USA 84, 6677-6681 (1987)).A λgt11 human placental cDNA library (Clonetech, Palo Alto, Calif.)provided the nucleic acid template. The LTA hydrolase encoding regionhad a length of about 1.9 kb. The amplified 1.9 kb DNA was isolated andcloned into the genomic baculovirus, Autographa californica nuclearpolyderosis virus (AcNPC) DNA, and the baculovirus expression vector wastransfected into Spodoptera frugiperda Sf-9 cells employing the calciumphosphate co-precipitation method (see, M. Summers, et al., Tex. Agric.Exp. Stn. Bull. 1555, 1-57 (1987). Recombinant LTA₄ hydrolase enzyme waspurified from the transfected Sf-9 cells essentially as described by J.Gierse, et al., supra.

One or more predetermined amounts of a compound of the invention wereincubated in assay buffer (0.1 M potassium phosphate, 5 mg/ml fatty acidfree BSA, 10% DMSO, Ph 7.4) for 10 minutes at room temperature with 250ng of recombinant hLTA₄ H to allow binding, if any, between the enzymeand inhibitor. The stock enzyme solution was 1 mg/m. LTA₄ hydrolase, 50Mm Tris, Ph 8.0, 150 Mm NaCl, 2.5 Mm beta-mercaptoethanol, 50% glycerol.The specific activity of the enzyme was about 650 Nmoles/min/mg. LTA₄(i.e., substrate) was prepared from the methyl ester of LTA₄ (Biomol,Inc., Plymouth Meeting, Pa.) by treating the methyl ester with 30 molarequivalents of LiOH at room temperature for 18 hours. The LTA₄ substratein its free acid form was kept frozen at -80° C. until needed. LTA₄(free acid) was thawed and diluted in assay buffer (minus DMSO) to aconcentration of 350 ng/ml and 25 μl (8 ng) of LTA4 substrate was addedto the reaction mixture (total volume of reaction mixture=200 μl at timezero. Each reaction was carried out at room temperature for 10 minutes.The reaction was stopped by diluting 25 μl of the reaction mixture with500 μl of the assay buffer without DMSO. LTA₄ was quantified in thediluted sample by a commercially available enzyme-linked immunoassayCaymen Chemical Col. Ann Arbor, Mich.! using the method recommended inthe manufacturer's instructions and compared to the amount of LTA₄produced in a negative control (i.e., essentially identical conditionsexcept without addition of an inhibitor compound). The IC₅₀ wasroutinely calculated from the data produced.

LTB₄ and Thromboxane Production by Calcium Ionophore Stimulated HumanBlood for LTB₄ Hydrolase Inhibitor Activity

Human blood, collected in heparin-containing Vacutainer tubes, wasdiluted 1:4 with RPMI-1640 media and 200 μl of the diluted blood wasadded into each of a 96-well microtiter plate. One or moreconcentrations of the leukotriene A₄ hydrolase inhibitor compounds beingtested were prepared (diluted in DMSO) and 2 μl added and gently mixedwith the diluted whole blood. After incubating for 15 minutes at 37° C.in a humidified incubator, calcium ionophore A13187 (Sigma Chemical Co.,St. Louis, Mo.) was added to a final concentration of 20 mcg/ml and theincubation continued under the same conditions for an additional 10minutes to allow LTB₄ formation. The reaction was terminated bycentrifugation (833 g, 10 minutes at 4° C.) and supernatant wereanalyzed for LTB₄ and thromboxane by commercially availableenzyme-linked immunoassays (Caymen Chemical Co., Ann Arbor, Mich.)according to the manufacturer's instructions. The IC₅₀ of each testcompound was determined from the amount of inhibition of LTB₄ productionas compared to an essentially identical assay in which no inhibitorcompound was present.

Ex Vivo LTB₄ and Thromboxane Production by Calcium Ionophore StimulatedMouse Blood for LTB₄ Hydrolase Inhibitor Activity

Leukotriene A₄ hydrolase inhibitor compounds of the present inventionwere diluted to a predetermined concentration in phosphate bufferedsaline containing 2% DMSO and 1% Tween 80. The compounds wereadministered by oral gavage to adult male outbred mice weighingapproximately 20-30 gm at a dose of 10 mg/kg body weight. (Compoundsgiven at a dose of 50 mg/kg body weight are designated in followingTable by the symbol, *) Sixty (60) minutes after administration of anLTA₄ inhibitor compound of the invention, blood was collected (intoheparin-containing tubes) from the retroorbital sinus. The heparinizedblood was added to the wells of a microtiter plate along with an equalvolume of RPMI-1640 media, and calcium ionophore A23187 was added to afinal concentration of 20 mcg/ml. The mixture was incubated for 10minutes at 37° C. in a humidified incubator. The reaction was terminatedby centrifugation (833 g. 10 minutes at 4° C.). Supernatant wereanalyzed for LTB₄ and thromboxane by commercially availableenzyme-linked immunoassays Caymen Chemical Co., Ann Arbor, Mich.! inaccordance with the manufacturer's instructions. The percent inhibitionwas determined by comparison to animals treated identically except thatthe solution administered by oral gavage was devoid of inhibitorcompound.

LTA₄ HYDROLASE INHIBITOR ACTIVITY

    ______________________________________                        Inhibition of                        Calcium           Recombinant  Ionophore- Murine Ex Vivo           Human LTA.sub.4                        induced LTB.sub.4                                   LTB.sub.4 Inhibition           Hydrolase    Production in                                   % I LTB.sub.4 /at 1           Assay        Human Blood                                   hour after           IC.sub.50    IC.sub.50  administration    Ex. #  (μM)      (μM)    of 10 mg/kg    ______________________________________    7      0.43         0.55       93%    8       0.0066      0.14       57%    10     0.59         0.55       83%    11     0.34         0.72       90%    12     --           0.22       87%    13     0.55         0.79       63%    14      <0.0005     0.19       78%    17     0.95         0.072      87%    18      0.027       0.19       94%    19     0.34         0.24       93%    ______________________________________     "--" means Not Determined

What is claimed is:
 1. A compound having the structure: ##STR49##wherein: A is ##STR50## wherein ..... represents a single or doublebond;q is 1 Y is --CH-- or --CH₂ -- B is --O--, --CH₂ -- or --CH₂ O-- nis 2 to 4 R¹ is H or C₁ to C₄ alkyl R² is (CH₂)_(m) R³ wherein m is 1 to3 R³ is CO₂ R⁴ R⁴ is H, alkyl, amino, alkylamino, dialkylamino; or NR¹R² is combined to form ##STR51## wherein r is 1 or 2, p is 0 to 3 and R³is as defined above.
 2. The compound of claim 1 wherein B is --O--. 3.The compound of claim 2 wherein NR¹ R² is combined to form ##STR52## 4.A pharmaceutical composition comprising compound having the structure:or a pharmaceutically acceptable salt or stereoisomer thereof, and apharmaceutically acceptable carrier, whereinA is ##STR53## wherein .....represents a single or double bondq is 1 Y is --CH-- or --CH₂ -- B is--O--, --CH₂ -- or --CH₂ O-- n is 2 to 4 R¹ is H or C₁ to C₄ alkyl R² is(CH₂)_(m) R³ wherein m is 1 to 3 R³ is CO₂ R⁴ R⁴ is H alkyl, amino,alkylamino, dialkylamino or NR¹ R² is combined to form ##STR54## whereinr is 1 or 2, p is 0 to 3 and R³ is as defined above.
 5. Thepharmaceutical composition of claim 4 wherein in the compound B is--O--.
 6. The pharmaceutical composition of claim 5 wherein in thecompound NR¹ R² is combined to form ##STR55##
 7. A method for treatingan LTB₄ -mediated inflammatory disease comprising administering to amammal in need of treatment a therapeutically effective amount of acompound having the structure: or a pharmaceutically acceptable salt orstereoisomer thereof, and a pharmaceutically acceptable carrier,whereinA is ##STR56## wherein ..... represents a single or double bondqis 1 Y is --CH-- or --CH₂ -- B is --O--, --CH₂ -- or --CH₂ O-- n is 2 to4 R¹ is H or C₁ to C₄ alkyl R² is (CH₂)_(m) R³ wherein m is 1 to 3 R³ isCO₂ R⁴ R⁴ is H alkyl, amino, alkylamino, dialkylamino or NR¹ R² iscombined to form ##STR57## wherein r is 1 or 2, p is 0 to 3 and R³ is asdefined above.
 8. The method of claim 7 wherein in the structure of thecompound B is --O--.
 9. The method of claim 8 wherein in the structureof the compound NR¹ R² is combined to form ##STR58##